the role of nibrin in doxorubicin-induced apoptosis and ...the role of nibrin in doxorubicin-induced...

The Role of Nibrin in Doxorubicin-Induced Apoptosis andCell Senescence in Nijmegen Breakage SyndromePatients LymphocytesOlga Alster1, Anna Bielak-Zmijewska1, Grazyna Mosieniak1, Maria Moreno-Villanueva2, Wioleta Dudka-

Ruszkowska3, Aleksandra Wojtala1, Monika Kusio-Kobiałka3, Zbigniew Korwek1, Alexander Burkle2,

Katarzyna Piwocka3, Jan K. Siwicki4, Ewa Sikora1*

1 Laboratory of the Molecular Bases of Aging, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland, 2Molecular Toxicology Group,

Department of Biology, University of Konstanz, Konstanz, Germany, 3 Laboratory of Cytometry, Nencki Institute of Experimental Biology, Polish Academy of Sciences,

Warsaw, Poland, 4Department of Immunology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland

Abstract

Nibrin plays an important role in the DNA damage response (DDR) and DNA repair. DDR is a crucial signaling pathway inapoptosis and senescence. To verify whether truncated nibrin (p70), causing Nijmegen Breakage Syndrome (NBS), isinvolved in DDR and cell fate upon DNA damage, we used two (S4 and S3R) spontaneously immortalized T cell lines fromNBS patients, with the founding mutation and a control cell line (L5). S4 and S3R cells have the same level of p70 nibrin,however p70 from S4 cells was able to form more complexes with ATM and BRCA1. Doxorubicin-induced DDR followed bycell senescence could only be observed in L5 and S4 cells, but not in the S3R ones. Furthermore the S3R cells onlyunderwent cell death, but not senescence after doxorubicin treatment. In contrary to doxorubicin treatment, cells from allthree cell lines were able to activate the DDR pathway after being exposed to c-radiation. Downregulation of nibrin innormal human vascular smooth muscle cells (VSMCs) did not prevent the activation of DDR and induction of senescence.Our results indicate that a substantially reduced level of nibrin or its truncated p70 form is sufficient to induce DNA-damagedependent senescence in VSMCs and S4 cells, respectively. In doxorubicin-treated S3R cells DDR activation was severelyimpaired, thus preventing the induction of senescence.

Citation: Alster O, Bielak-Zmijewska A, Mosieniak G, Moreno-Villanueva M, Dudka-Ruszkowska W, et al. (2014) The Role of Nibrin in Doxorubicin-InducedApoptosis and Cell Senescence in Nijmegen Breakage Syndrome Patients Lymphocytes. PLoS ONE 9(8): e104964. doi:10.1371/journal.pone.0104964

Editor: Arianna L. Kim, Columbia University Medical Center, United States of America

Received March 26, 2014; Accepted July 16, 2014; Published August 13, 2014

Copyright: � 2014 Alster et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. Relevant data are included within the paper.

Funding: This work was supported by National Center of Science in Poland (grants: 2011/01/M/NZ1/01597, UMO-2011/01/B/NZ3/02137 and 0728/B/P01/2011/40). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* Email: [email protected]

Introduction

Nijmegen Breakage Syndrome (NBS) is a rare autosomal

recessive disorder characterized by genomic instability and

increased risk of haematopoietic malignancies observed in more

than 40% of the patients by the time they are 20 years old [1].

NBS is caused by mutations in the NBN gene (originally

designated as NBS1) encoding nibrin. More than 90% of the

patients are homozygous for the same mutation (c.657-661del5)

what results in the formation of two truncated fragments of the

95 kDa nibrin: 26 kDa N-terminal fragment (p26-nibrin) and

70 kDa C-terminal fragment (p70-nibrin), which are produced by

alternative initiation of translation at a cryptic upstream start

codon. This mutation is actually hypomorphic as the truncated

p70-nibrin is able to retain some of the vital cellular functions of

the full-length protein. The truncated p70-nibrin can form the

MRN (Mre11-Rad50-Nbs1) complex with two other proteins,

Mre11 and Rad50 [2,3]. However, null mutation of the Nbn gene

is lethal in mice [4].

Stress-induced premature senescence (SIPS) is a relatively fast,

telomere erosion independent, process. Among its characteristic

features we can distinguish irreversible growth arrest, altered cell

morphology, DNA foci formation, activation of senescence-

associated b-galactosidase (SA-b-Gal) and senescence associated

secretory phenotype-SASP (reviewed in [5]). Recently, it was

shown that double-strand DNA breaks (DSBs), after induction of

the DNA damage response (DDR), are crucial for cellular

senescence [6]. Briefly, upon DSB induction ataxia telangiectasia

mutated (ATM) kinase is activated. The activated kinase

phosphorylates nibrin at its Ser 343 residue and H2AX histone,

at its Ser 139 residue (cH2AX). Phosphorylated nibrin forms a

trimeric complex (MRN) along with Mre11 and Rad50, which is

recruited to the vicinity of DSBs where nibrin interacts with

cH2AX [7]. Ultimately, Chk1, Chk2 (checkpoint kinase 1 and 2,

respectively) and p53 are activated. p53 promotes senescence

(when DNA damage is irreparable) via transactivation of

CDKN1A, which encodes the cyclin dependent kinase inhibitor

p21 [5].

DDR activation, not only can lead to senescence but also to

transient cell cycle arrest and DNA repair or apoptosis.

Improperly functioning DDR often results in increased radiosen-

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sitivity, genomic instability and cancer development. Since NBS1

deficient cells are characterized by genomic instability and NBS

patients suffer from haematopoietic malignancies, we hypothesized

that the molecular pathways leading to DNA damage-induced

senescence might be impaired in patients affected with this disease.

Most cell lines derived from NBS patients were established

following transformation with viral oncogenes, which inhibit key

regulatory genes such as the tumor suppressor gene proteins p53

and pRb, thus allowing the cell to bypass the senescence program

and become immortal [8]. Accordingly, spontaneously immortal-

ized T cell lines, S3R and S4, carrying the same mutation within

the NBN gene, but with a seemingly functional p53/p21 response

after gamma irradiation [9], are a very useful cellular model in

studying the mechanisms of DNA damage-induced senescence.

Therefore we used two cell lines derived from NBS patients (S3R

and S4) and the control, L5 cell line (spontaneously immortalized

spleenocytes obtained from a healthy donor) to examine if they are

prone to DNA damage-induced senescence. To induce DNA

damage and DDR activation we used doxorubicin, which is a

DNA damaging agent acting through different mechanisms. It can

lead to the formation of direct and indirect DNA damage through:

intercalation into DNA, DNA binding and alkylation, DNA cross-

linking, interference with DNA unwinding or DNA strand

separation, helicase activity as well as inhibition of topoisomerase

II and generation of free radicals [10].

Materials and Methods

1. Cell linesThe spontaneously immortalized T cell lines: S3R and S4 were

established from peripheral blood mononuclear cells (PBMC)

derived from NBS patients homozygous for the 657del5 mutation

of the NBN gene [9] and the L5 cell line was established from the

spleen of a healthy donor as described previously [9,11]. All of the

cell lines were cultured in the RPMI 1640 medium (Gibco, Life

Technologies, Warsaw, Poland) supplemented with 10% FCS

(Biochrom, Biomibo, Warsaw, Poland), 50 mg/ml gentamycin

(Sigma, Poznan, Poland), 2 mM glutamine (Sigma, Poznan,

Poland) and 20 U/ml of IL-2 (R&D, Biokom, Warsaw, Poland).

Human vascular smooth muscle cells (VSMCs) were obtained

from Lonza (Basel, Switzerland). hVSMC were grown in SmBM

medium (Lonza, Basel, Switzerland). S3R, S4 and L5 cells were

seeded at a density of 0,26106/ml 24 h before doxorubicin

(Sigma, Warsaw, Poland) treatment. VSMCs were seeded at a

density of 26103/cm2 24 h before transfection.

2. DNA content and cell cycle analysisFor DNA analysis the cells were fixed in 70% ethanol and

stained with PI solution (3,8 mM sodium citrate, 50 mg/ml RNAse

A, 500 mg/ml PI in PBS). All of the used agents were purchased at

Sigma Aldrich (Poznan, Poland). DNA content was assessed using

flow cytometry and analyzed with the CellQuest Software. 10000

events were collected per sample (FACSCalibur, Becton Dick-

inson, Warsaw, Poland).

3. ImmunoprecipitationS3R and S4 cells were lysed with modified RIPA buffer [12].

Equal amounts of protein (750 mg) were taken for immunopre-

cipitation. The supernatants were precleared by adding Protein A/

G PLUS-Agarose Immunoprecipitation Reagent (Santa Cruz

Biotechnology, Inc., Dallas, Texas, USA) and incubated with IP

antibody-IP matrix complexes overnight using NBS1 rabbit

polyclonal antibody (Sigma Aldrich, Poznan, Poland), Mre11

rabbit monoclonal antibody (Cell Signaling, Lab-JOT Ltd.,

Warsaw, Poland), ATM rabbit monoclonal antibody (Epitomics,

Burlingame, California, USA) according to the manufacturer’

protocol (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA).

Beads were washed with PBS and immune complexes were eluted

with sodium dodecyl sulphate (SDS)-containing buffer and boiled.

Mre11, BRCA1, ATM and NBS1 were detected using the

Western blotting technique with the following antibodies: BRCA1

mouse monoclonal (R&D, Biokom, Warsaw, Poland), ATM rabbit

monoclonal antibody (Epitomics, Burlingame, California, USA),

Mre11 rabbit monoclonal antibody (Cell Signaling, Lab-JOT Ltd.,

Warsaw, Poland), NBS1 rabbit polyclonal antibody (Sigma

Aldrich, Poznan, Poland) and secondary rabbit polyclonal

antibody conjugated with horseradish peroxidase (Dako, Poland).

4. Western blotting analysisWhole cell protein extracts were prepared according to the

Laemmli method [13]. Equal amounts of protein were separated

electrophorectically in 8, 12 or 15% SDS-polyacrylamide gels and

afterwards transferred to nitrocellulose membranes. Membranes

were blocked in 5% non-fat milk dissolved in TBS containing

0,1% Tween-20 (Sigma Aldrich, Poznan, Poland) for 1 h at RT

and incubated with one of the primary monoclonal or polyclonal

antibodies: anti-ATM (1:500) (Millipore, Merck, Warsaw, Poland),

anti-p-ATM Ser 1981 (1:1000), H2AX (1:500) and anti-cH2AX

(1:1000) (Abcam, Cambridge, UK), anti-p16 (1:500), anti-p53

(DO-1) (1:500), anti-p21 (C-19) (1:500) (Santa Cruz Biotechnology

Inc., Dallas, Texas, USA), anti-p-p53 Ser 15, anti-Chk1, anti-p-

Chk1 Ser 317, anti-Chk2, anti-p-Chk2 Thr 68, anti-NBS1 Ser 343

(Cell Signaling, Lab-JOT Ltd., Warsaw, Poland), anti-PARP1

(1:1000) (Becton Dickinson, Diag-med, Warsaw, Poland) anti-

NBS1 (1:500), anti-b-actin (1:50000) (Sigma Aldrich, Poznan,

Poland) and anti-GAPDH (1:50000) (Millipore, Merck, Warsaw,

Poland). The proteins were detected with appropriate secondary

antibodies conjugated with horseradish peroxidase and ECL

reagents (GE Healthcare, Buckinghamshire, UK), according to the

manufacturer’s protocol.

5. Gamma irradiation procedureAsynchronously growing cells were treated with 4 Gy of c-

irradiation. Immediately after irradiation the cells were diluted to a

concentration 0,256106 cells/ml and cultured for 3 h. Cells were

collected after 3 h (untreated and treated with 4 Gy of irradiation).

Whole cell extracts were prepared for Western blotting analysis.

6. Silencing of the NBN geneTo downregulate NBN expression the cells were seeded in 6 or

12-well plates (26104 or 86103 cells per well, respectively) and

transfected with 60 nM siRNA (NBN or negative) (Life Technol-

ogies, Warsaw, Poland) using Lipofectamine 2000 (Life Technol-

ogies, Warsaw, Poland). Transfection was performed according to

the manufacturer’s protocol. About 20 h after transfection

medium was replaced with fresh one and cells were cultured for

three days in the presence of doxorubicin (100 nM) (Sigma

Aldrich, Poznan, Poland).

7. Detection of SA-b-GalDetection of SA-b-Gal was performed according to Dimri et al.

(1995) [14]. Briefly, cells were fixed with 2% formaldehyde, 0,2%

glutaraldehyde in PBS, washed and exposed overnight at 37uC to

a solution containing: 1 mg/ml 5-bromo-4-chloro-3-indolyl-b-D-

galactopyranoside, 5 mM potassium ferrocyanide, 5 mM potassi-

um ferrycyanide, 150 mM NaCl, 2 mM MgCl2 and 0,1 M

phosphate buffer, pH 6,0. All of the used agents were purchased at

Apoptosis and Cell Senescence in Nijmegen Breakage Syndrome Patients


Sigma Aldrich (Poznan, Poland). Photos were taken using the

Evolutions VF digital CCD camera (Media Cybernetics, Rock-

ville, Maryland, USA).

8. Apoptosis detectionThe level of apoptosis was measured by flow cytometry

(FACSCalibur) using the annexin V/7-AAD assay (Becton

Dickinson, Diag-med, Warsaw, Poland). Externalization of phos-

phatidylserine (PS) to the outer layer of the cell membrane was

examined by binding of annexin V in the presence of 7-AAD, a

dye which stains dead cells. Briefly, cells were washed with PBS,

suspended in the annexin V binding buffer, stained for 15 min

with annexin V conjugated with PE and 7-AAD. Analysis was

performed with FACSCalibur using the CellQuest Software (BD

Biosciences, Warsaw, Poland). 10000 events were collected per

sample.

9. Bromodeoxyuridine labeling assayTo evaluate DNA synthesis BrdU (Sigma Aldrich, Poznan,

Poland) was added to the medium (10 mM) and cells were cultured

for 24 h. Afterwards the cells were fixed in ethanol. BrdU was

detected using a primary antibody against BrdU (Becton

Dickinson, Warsaw, Poland) and a secondary Alexa 488 antibody

(Life Technology, Warsaw, Poland). The cells were observed

under a fluorescence microscope (Nikon, Tokyo, Japan) with the

use of 450–490 nm -excitation wavelength. Photos were taken

using the Evolutions VF digital CCD camera (Media Cybernetics,

Rockville, Maryland, USA).

10. ImmunocytochemistryFor immunofluorescence the cells were fixed with 2% parafor-

maldehyde (Sigma Aldrich, Poznan, Poland) at RT for 20 minutes

and afterwards were incubated on slides with the anti-53BP1

monoclonal antibody (Novus, Cambridge, USA). Secondary anti-

rabbit Alexa 488-conjugated IgG antibody was used (Life

Technology, Warsaw, Poland). Cells were observed under a

fluorescence microscope (Nikon, Tokyo, Japan) and photos were

taken using the Evolutions VF digital CCD camera (Media

Cybernetics, Rockville, Maryland, USA).

11. Fluorimetric Detection of DNA unwinding (FADU)methodA modified and automated version of the FADU (Fluorimetric

Detection of alkaline DNA Unwinding) method was used to

measure the percentage of double-stranded DNA after treatment

with doxorubicin (1 and 10 mM). The percentage of DNA damage

was analyzed 30, 60 and 90 min after treatment with a DNA

damaging agent as described previously by Moreno-Villanueva et

al. [15,16]. The method is based on partial denaturation

‘‘unwinding’’ of double-stranded DNA under controlled alkaline

and temperature conditions. DNA strand breaks are sites where

DNA unwinding can start. Briefly, after infliction of DNA damage

cell lysis was performed. DNA unwinding was terminated by

adding a neutralization solution. SybrGreen, a commercially

available dye, which only binds to double stranded DNA, was used

to determine the amount of double-stranded DNA. The lower the

fluorescence the less double-stranded DNA in the sample.

12. Statistical analysisStudent’s T test was used to calculate statistical significance: *,

p,0,05; **, p,0,01; ***, p,0,001.

Results

1. FADU analysis of L5, S3R and S4 cells treated withdoxorubicinDoxorubicin is a DNA-damaging agent which is widely used in

chemotheraphy. It has been shown that cytostatic doses of

doxorubicin can lead to the induction of cellular senescence. Cells

sensitivity to this agent can vary between different types of cells.

Therefore the first step was to analyze the cells sensitivity to

treatment with this agent. To do this we used the FADU method.

FADU enables to measure, in an automatic way, the percentage of

double-stranded DNA [15,16], which accounts for 100% in

control cells (Fig. 1). SybrGreen, a fluorescent dye used in this

method, binds only to double-stranded DNA. Therefore, the less

intensive fluorescence the less double-stranded DNA can be

observed. To this end we treated all of the cell lines: with the

mutated form of nibrin (S3R and S4) and spontaneously

immortalized cells from a healthy donor (L5) with two concen-

trations of doxorubicin (1 and 10 mM) and analyzed the

percentage of double-stranded DNA, after short periods of time

(30, 60 and 90 min). The most sensitive, to treatment with

doxorubicin, were the S3R cells. In the case of this cell line, a

significantly lower amount of double-stranded DNA could be

found at all of the analyzed time points after treatment with both

concentrations of doxorubicin, in comparison with the untreated

cells. In the case of S4 cell line a statistically significant decrease of

the percentage of double-stranded DNA, in comparison with

control cells, could be observed 90 min after treatment with the

lower (1 mM) concentration of doxorubicin and in all of the time

points after treatment with the higher (10 mM) concentration of

this agent. This shows that even though S3R and S4 cell lines

possess the same NBN mutation their sensitivity to treatment with

doxorubicin is different. Furthermore, it turned out that, the

control (L5) cells are more sensitive to doxorubicin treatment than

the S4 cells, however less sensitive than the S3R cells. The

obtained results allowed us to speculate that different concentra-

tions of doxorubicin could be cytostatic for particular cell lines and

different doses could be needed for the induction of doxorubicin-

induced senescence.

2. Cell cycle arrest and apoptosis in doxorubicin-treatedL5, S3R and S4 cellsOne of the hallmarks of senescence is cell cycle arrest. Cells

undergoing senescence can be arrested in the G1/S or G2/M

phases of the cell cycle, however stress-induced premature

senescence (SIPS) is predominantly associated with cell cycle

arrest in the G2/M phase of the cell cycle. We treated L5, S3R

and S4 cells with various concentrations of doxorubicin, ranging

from 10 to 250 nM and analyzed DNA content using flow

cytometry. As it is shown in Figure 2A and in Table 1 treatment

with doxorubicin arrested cells from all of the cell lines in the G2/

M phase of the cell cycle. In case of the control (L5) cell line the

majority of cells were arrested after treatment with 50 nM

doxorubicin (approximately 30%). The largest fraction of S4 cells

(almost 50%) arrested in the G2/M, was observed after treatment

with 100 nM doxorubicin. In the S3R cell population the majority

of cells were found in the G2/M phase of the cell cycle after

treatment with 10 and 50 nM doxorubicin (about 35%). The

subG1 fraction which represents apoptotic cells did not exceed

11% in the case of the L5 cell line and 12% in the case of S4 cells.

S3R cells were much more prone to spontaneous apoptosis and

about 30% of the cells were found in the subG1 fraction. A

concentration dependent increase in the level of apoptosis could be

observed after treatment with doxorubicin in all of the cell lines.



The DNA content analysis can underestimate the level of

apoptosis due to the fact that cells with 4C DNA undergoing

apoptosis, may have $2C DNA and cannot be distinguished from

the cells found in the S and G1 phases of the cell cycle. Therefore,

to estimate more accurately the percentage of cells undergoing

apoptosis, we performed the Annexin V/7-AAD cytometric

analysis. As expected this method revealed more apoptotic cells

in all of the analyzed cell lines in comparison with the DNA

content analysis. However, in the S4 cells, concentration

dependence after treatment with doxorubicin still could not be

observed. In the case of S3R cells about half of the cell population

underwent cell death after treatment with 50 and 100 nM

doxorubicin, i.e. significantly more than control cells (Fig. 2B).

These results show that S3R cells are very prone to both

spontaneous and doxorubicin induced apoptosis and generally

more sensitive to the treatment than the S4 cells. Nonetheless, a

substantial fraction of cells from all of the cell lines can be arrested

in the G2/M phase of the cell cycle upon treatment with different

concentrations of doxorubicin.

Since DNA-damage induced senescence is associated with

persistant activation of the DNA-damage response (DDR)

pathway, which can be observed 24–48 h after treating the cells

with a DNA damage inducing agent, we decided to analyze the

activation of this pathway after treating the cells with the selected

concentrations of doxorubicin. Such concentrations of doxorubi-

cin were selected which led to the accumulation of the most cells in

the G2/M phase of the cell cycle and relatively low level of cell

death. S3R cells were treated with 10 nM and S4 cells with

100 nM doxorubicin. For comparison we used spontaneously

immortalized spleenocytes obtained from a healthy donor (L5),

treated with 50 nM doxorubicin (Fig. 2C). In the case of the L5

cell line, we observed the presence of p-ATM (Ser 1981), p-p53

(Ser 15) and p-Chk1 (Ser 317), even in untreated cells. After

treatment with doxorubicn (24 h) increased levels of these proteins

and the presence of Chk2 and p-Chk2 (Thr 68) was observed

proving the presence of an active DDR. In untreated S4 cells the

phosphorylated form of Chk1 (Chk1 Ser 317) was detected.

Interestingly, after treatment with doxorubicin we noticed a

significant increase in the level of p-ATM (Ser 1981), p-Chk1 (Ser

317), p-Chk2 (Thr 68), p-p53 (Ser 15) and cH2AX. Our results

show that upon treatment with doxorubicin the DDR pathway is

only activated in the L5 and S4 cell lines, however this process

can’t be observed in the S3R cell line.

3. Doxorubicin-induced senescence of L5 and S4, but notS3R cellsThere is data showing that immortalized and cancer cells retain

the ability to undergo senescence including that induced by DNA

damage [17,18]. We have previously shown [19] that treatment of

human colon cancer HCT116 cells with a low dose of doxorubicin

for one day, followed by culture in a drug-free medium, led to the

induction of senescence. Therefore, we decided to use the same

experimental approach and treated the L5, S3R and S4 cells with

the chosen concentrations of doxorubicin (50 nM for L5, 10 nM

for S3R and 100 nM for S4) for 24 hours and afterwards cultured

the cells for four days in a drug free medium (1+4). We observed a

time-dependent increase in the number of SA-b-Gal positive cells

in L5 and S4, but not in S3R cell line (Fig. 3A, B). In case of the

L5 cell line the majority of SA-b-Gal-positive cells (approximately

95%) were observed on day 1+4. In case of the S4 cell line the

most SA-b-Gal positive cells were observed on day 1+3(approximately 50%). The presence of SA-b-Gal positive cells

was accompanied by an increase in the level of p53 (Ser 15) in

both cell lines, however a time dependent increase in the level of

p21 was only observed in the S4 cell line. Surprisingly, in the L5

cell line, a time dependent decrease in the level of this protein was

found (Fig. 3C). Two crucial pathways play an important role in

senescence: p53-p21 and p16-pRb. Sometimes these pathways

overlap therefore we also decided to check the level of p16, which

is a key protein in the p16-pRb pathway.

Figure 1. FADU analysis in L5, S3R and S4 cells treated withdoxorubicin. The percentage of double-stranded DNA, was measuredin all of the cell lines using the FADU method. All of the cell lines weretreated with 1 and 10 mM doxorubicin and the measurements wereperformed 30, 60 and 90 min after treatment with this agent. Data iscalculated as the percentage of control. The values are means 6 SDobtained from three independent experiments. Statistical significancewas estimated using the Student’s T test.doi:10.1371/journal.pone.0104964.g001



Figure 2. Dose-dependent influence of doxorubicin on cell cycle arrest, apoptosis and activation of the DNA damage response(DDR). A. DNA content, analyzed by flow cytometry, 24 h after treatment with different concentrations of doxorubicin (0–250 nM). Representative



The p16-mediated senescence acts mainly through the retino-

blastoma (pRb) pathway by inhibiting the action of the cyclin

dependent kinases and leads to G1 cell cycle arrest [20]. We did

not observe any changes in the level of this protein in the S3R and

S4 cell lines, however, a time dependent decrease in the level of

p16 was observed in the L5 cell line (Fig. 3C). The observation

made in the L5 cell line requires further elucidation. It seems that

untreated S3R cells might have the p53/p21 pathway already

active, which is further slightly activated after treatment with

doxorubicin, but this is not accompanied by an increase in SA-b-Gal activity. In the L5 and S4 cells stronger activation of the p53/

p21 pathway correlated with an increase in SA-b-Gal activity.

This encouraged us to investigate whether the lack of induction of

senescence in the S3R cell line was due to the fact that the cells

underwent cell death. Using the annexin V/7-AAD assay, we

measured the level of apoptosis a day after treatment with

doxorubicin and on subsequent days after transferring the cells to

fresh medium (Fig. 3D). In all of the cell lines we observed a time-

dependent increase in the level of apoptosis. Three days after

culturing the cells in drug free medium (1+3) approximately 55%

of cells underwent apoptosis in all of the cell lines, however it

should be underlined that the cells were treated with different

concentrations of doxorubicin. The S4 cells were treated with a

ten times higher concentration of doxorubicin (100 nM) than the

S3R cells (10 nM). Moreover, in the case of the L5 and S3R cell

lines a high basal level of apoptosis could be observed. Despite the

high level of cell death, a fraction of S4 (more than 40% of SA-b-Gal positive cells on day 1+4) and L5 (about 95% of SA-b-Gal

positive cells on day 1+4) of cells, that survived, were able to

undergo senescence.

4. The level of p70-nibrin in S3R and S4 cellsWe were interested whether the differences in the cells

susceptibility to doxorubicin treatment and cell fate were due to

a different level of the truncated form of nibrin (p70), which is

present in the S3R and S4 cells. To elucidate this we performed an

immunoprecipitation assay which showed the same level of p70-

nibrin in both untreated and doxorubicin treated S3R and S4 cells

(Fig. 4A). The p95 form of nibrin was not detected neither in S3R

nor in S4 cells, however it was observed in VSMC cells, which

were used as a positive control. To confirm the above observation

and to exclude the possibility that the amount of immunoprecip-

itated protein was not equal, we verified the p70-nibrin level also

after IP, using the anti-Mre11 antibody. In this case we checked

the level of Mre11 by WB, as a loading control, and followed with

analysis of nibrin. Also this time we did not detect any differences

histograms from one of three independent experiments. B. Concentration-dependent apoptosis measured 24 h after treatment with doxorubicin (0–250 nM). The percentage of apoptotic cells was estimated by the Annexin V/7-AAD flow cytometry assay in three independent experiments. The barsshow means 6 SD values. Data was analyzed using the CellQuest software. Statistical significance was estimated using the Student’s T test. C.Expression of the DDR proteins analyzed by Western blotting in control (C) and treated with doxorubicin (D) S3R, S4 and L5 cells. Whole cell extractswere prepared 24 h after cell treatment with the following cytostatic concentrations of dox: 50 nM (L5), 10 nM (S3R), 100 nM (S4) and b-actin wasused as a loading control.doi:10.1371/journal.pone.0104964.g002

Table 1. DNA content (%) in L5, S3R and S4 cells treated with different concentration of doxorubicin.

L5

% untreated 10 nM 50 nM 100 nM 250 nM

SubG1 11,4765,32 7,1865,85 13,8365,45 20,4865,42 24,6469,37

G1 59,5765,61 65,1963,24 49,6764,6 57,49610,15 59,2865,42

S 7,4964,86 6,2763,92 3,9260,21 2,9260,92 3,3360,24

G2/M 19,5263,46 21,7365,98 30,1611,14 17,9566,36 11,7362,99

S3R

% untreated 10 nM 50 nM 100 nM 250 nM

SubG1 27,9364,14 29,2365,9 31,6465,49 33,6365,23 32,6461,28

G1 25,8364,1 19,0463,9 17,1864,01 19,7764,56 25,8762,45

S 15,5763,72 12,5361,12 10,761,22 15,2861,12 16,0160,59

G2/M 25,0862,09 34,8464,49 35,2464,4 26,0763,57 1965,57

S4

untreated 10 nM 50 nM 100 nM 250 nM 500 nM

SubG1 6,4364,87 7,5964,07 6,5662,74 6,7261,57 11,3763,39 10,6760,73

G1 42,01615,9 40,37612,17 31,6369,31 20,0768,12 18,1562,18 35,5361,49

S 17,1365,69 16,6765,85 14,1265,28 1164,73 7,6863,56 20,7864,08

G2/M 20,866,52 24,1166,18 35,5465,18 48,4865,64 39,7969,42 31,0265,35

The cells were treated with doxorubicin for 24 h and DNA content was measured by flow cytometry. The percentage of DNA in all phases (including subG1) of the cellcycle is shown. Data obtained after treatment with the selected concentrations of doxorubicin, chosen for the induction of senescence, is shown in bold.doi:10.1371/journal.pone.0104964.t001



Figure 3. The induction of cellular senescence and apoptosis upon doxorubicin treatment. A. SA-b-Gal activity. Cells were treated for 1day with doxorubicin (L5 - 50 nM, S3R - 10 nM and S4 - 100 nM) and then cultured in doxorubicin-free medium (1+n; n- are days of culture withoutdoxorubicin). The bars show means 6 SD. The percentage of SA-b-Gal positive cells from at least three independent experiments and representativeimages from one of three independent experiments. B. Magnification 200x. C. Expression of protein markers of senescence (p-p53, p53, p21 and p16)analyzed by Western blotting in untreated (0) cells and on the following days after culturing in doxorubicin-free medium (1+n), b-actin was used as aloading control. D. The percentage of apoptotic cells after treatment with doxorubicin estimated by the AnnexinV/7-AAD flow cytometry assay. Thebars show means 6 SD values. Data were obtained from three independent experiments. Statistical significance was estimated using the Student’s Ttest.doi:10.1371/journal.pone.0104964.g003



in the level of p70-nibrin. To verify the functionality of the

truncated nibrin we analyzed its binding to ATM. After

immunoprecipitating either ATM or nibrin it was observed that

p70-nibrin was able to form a complex with ATM in both NBS1

deficient cell lines (Fig. 4B). However, the IP revealed that more

ATM immunoprecipitated with p70-nibrin in S4 than S3R cells.

This may suggest that formation of DNA damage-induced ATM-

nibrin complex is more efficient in S4 cells. This difference was

already found in untreated cells and correlated with the observed

higher phosphorylation of ATM in response to doxorubicin

treatment of S4 cells (Fig. 2C). The possible better function of the

DNA damage/repair response in S4 cells was confirmed by a

further IP experiment showing that in these cells more BRCA1

was immunoprecipitated with ATM (Fig. 4C) suggesting that S4

are more efficient in DNA repair than the S3R cells.

5. Radiation induced activation of the DDR pathway inL5, S3R and S4 cellsDespite the fact that Nijmegen Breakage Syndrome was caused

by the same mutation in the S3R and S4 cell lines their

susceptibility to doxorubicin treatment differed. To verify whether

this was a characteristic feature of only doxorubicin, we used a

different DNA-damaging agent. Therefore the cells ability to

activate the DDR pathway after being exposed to c-radiation (4Gy

and cultured for 3 h) was analyzed (Fig. 5). Interestingly, exposure

to c-radiation of both S4 and S3R as well as control (L5) cells led

to an efficient induction of DDR. An increase in the level of the

following proteins was observed in all of the analyzed cell lines: p-

ATM (Ser 1981), p-Chk1 (Ser 317), p-p53 (Ser 15) and cH2AX.

The phosphorylated form of Chk2 (Thr 68) was only noticed upon

exposure to c-radiation of the S4 cells. This indicated that these

cells retain the capacity to upregulate the components of the DDR

pathway, at least for a short period of time.

6. The role of nibrin in DNA damage induced senescenceof human Vascular Smooth Muscle CellsSince a relatively low level of the truncated nibrin (p70-nibrin)

in S4 cells was sufficient for activation of the DDR signaling

pathway followed by senescence, we asked whether downregula-

tion of the NBS1 protein in normal cells would influence DDR

activation and senescence upon treatment with doxorubicin.

Transfection of L5 cells, using the nucleofection method, turned

out to be unsuccessful. Only 25% of the transfected cells were

viable 24 h after transfection. Therefore to analyze the effect of

downregulation of nibrin on the induction of senescence, we used

vascular smooth muscle cells (VSMCs) which were shown by us to

undergo senescence after treatment with doxorubicin [21]. Before

treatment with doxorubicin, the cells were transfected with

negative siRNA or NBN siRNA with 85% transfection efficiency

measured a day after transfection (not shown). As shown in

Figure 6A the level of NBS1 in cells transfected with NBN siRNA

and cultured in the presence of doxorubicin for three days was

reduced from two to four times. Moreover the levels of p-NBS1

(Ser 343) and p-ATM (Ser 1981) were substantially reduced in

these cells. However, there were no differences in the level of p53

and p21 proteins between cells transfected with negative siRNA

and NBN siRNA (Fig. 6A). Next we decided to verify whether the

downregulation of nibrin would affect the formation of 53BP1 foci,

after treatment with doxorubicin. Recently 53BP1 has been

recognized as a convenient marker of DSBs [22]. We observed

that the formation of 53BP1 foci was not affected when the level of

NBS1 was reduced (Fig. 6B, 6C). This could suggest that

senescence was also not affected in cells with reduced level of

NBS1. Indeed, the percentage of SA-b-Gal positive cells was

substantially increased already two days after treatment with

doxorubicin in both types of cells and accounted for 100% on day

3 of treatment with doxorubicin (Fig. 6D, 6E). These results were

confirmed using the BrdU incorporation assay, which showed

complete inhibition of proliferation in cells which were transfected

with negative siRNA and NBN siRNA and subsequently treated

with doxorubicin (Fig. 6F).

Taken together, the obtained results performed on human

VSMCs indicate that a substantially reduced level of NBS1 did not

influence doxorubicin-induced DDR and senescence in these cells.

Discussion

The aim of our study was to investigate the role of nibrin in

doxorubicininduced senescence.

Cellular senescence is associated withpermanent growth arrest.

We can distinguish two types of cellular senescence: replicative

which is telomere shortening dependent and stress-induced

premature senescence, which is telomere shortening independent.

Replicatively senescing cells are believed to activate the G1

restriction point. However, it was recently documented that

replicative senescence can stop the cells in both the G1/S and G2/

M phases of the cell cycle [23] while SIPS is mainly associated with

cell cycle arrest cells in the G2/M phase of the cell cycle.

NBS1 deficient cells have improperly functioning cell cycle

checkpoints [24], including a defect of the DNA damage induced

intra-S-phase checkpoint which is responsible for the radio-

resistant DNA synthesis (RDS)- a continuation of DNA synthesis

despite the presence of radiation-induced DNA damage [25].

However, the reports concerning the status of cell cycle

checkpoints in NBS deficient cells are discrepant, since both

impaired and normal G1/S or G2/M arrest after cell irradiation

have been reported (reviewed by [26]). Previously it was

documented that S3R cells had a reduced capacity to undergo

G1 arrest and showed a marked accumulation of cells in the G2/

M phase of the cell cycle 24 h after 4Gy c-irradiation, though to a

lesser extent than the S4 cells [27]. We have shown that treatment

with doxorubicin of L5, S3R and S4 cells led to an arrest in the

G2/M phase of the cell cycle. However, we proved that S3R cells

had a less efficient G2 checkpoint than S4 cells. Treatment of S3R

cells with 100 nM doxorubicin, the concentration which halted

most of the S4 cells in the G2/M phase of the cell cycle, led to

massive cell death of S3R cells. Nevertheless the percentage of

S3R cells which were arrested in the G2/M phase of the cell cycle,

after treatment with the selected, cytostatic concentrations of

doxorubicin, was comparable to the one observed in control L5

cells.

The higher propensity of S3R than S4 cells to undergo

apoptosis was connected with a decrease in the level of double-

stranded DNA as revealed using the FADU method. One should

keep in mind that doxorubicin is a DNA-damaging agent which

acts through different mechanisms. Amongst all, it induces the

formation of cross-links which prevent DNA from unwinding. The

FADU method enables to measure DNA susceptibility to unwind,

which is a function of the number of chromatin modifications.

Therefore the FADU method, in the context of this particular

agent, can only be used as a screening method which enables to

verify the cells susceptibility to treatment with different concen-

trations of doxorubicin. Nevertheless, the decrease in the amount

of double-stranded DNA was observed with the increasing

concentrations of doxorubicin and time of treatment in all of the

three examined cell lines, proving that at least a portion of DNA

acquires double strand breaks upon treatment with doxorubicin.



Generally, NBS1 deficient cells have impaired DNA repair. This

process seems to be more severe in the S3R than in the S4 cells

due to the lower level of the BRCA1 protein, which doesn’t

interact with ATM in the S3R cells. It was reported that

downregulation of the NBS1 protein level by siRNA led to an

increase in irradiation-induced mutation frequency in human

lymphoblastoid cells [26]. Moreover, it is worth to note that null

mutation of Nbs is lethal in mice [4].

Interestingly, the presence of less double-stranded DNA, after

treatment with doxorubicin, in the S3R cells, than in the S4 and

L5 cells, was not linked to ATM activation. However, we observed

increased levels of p-ATM and its downstream targets such as p-

Chk1, p-p53 and cH2AX 24 h after treatment with doxorubicin

in control (L5) and S4 cells. Moreover in the S4 cells upon

doxorubicin treatment, a substantial increase in the level of p-

Chk2 (Thr 68) could be seen. Surprisingly all of the cell lines

retained the ability to activate DDR upon exposure to c-radiation.Several studies showed severe impairment of the DDR activation

in NBS1 deficient cells. Namely, cells from NBS patients have

been reported to be deficient in ATM phosphorylation of p53,

Chk2 and other substrates following DNA damage. Other studies

showed that the C-terminal fragment of nibrin was sufficient to

stimulate ATM activation at early times after irradiation. In

contrast, nuclear expression of a nibrin transgene lacking the C-

terminal 100 amino acids was unable to stimulate ATM activation

under the same conditions ([28] and literature there). This was

most likely due to the lack of the ATM binding domain. We have

shown that despite the presence of the same NBN gene mutation,

DDR is only activated in the S4 cells. Furthermore, this pathway

was also activated in the L5 cells. In S3R cells some elements of

the DDR (p-p53, p-Chk1 and p-Chk2) were already present in

untreated cells and 24 h treatment with doxorubicin did not lead

to an increase in the level of these proteins. It is tempting to

speculate that the different response of the two NBS1 deficient cell

lines to treatment with doxorubicin is caused by the presence of a

lower level and/or nonfunctional truncated form of nibrin (p70-

nibrin) in S3R cells. Indeed, it has been shown that the level of

p70-nibrin can vary in cells obtained from NBS patients [2].

However, our results showed the same amount of p70-nibrin in S4

and S3R cells. Moreover, in both cell lines p70-nibrin co-

immunoprecipitated with ATM. Nevertheless we observed that a

higher level of p70-nibrin precipitated with ATM in S4 cells than

in the S3R cells. In contrast to the results obtained using the S4

cells and the L5 cells with wild-type NBN gene, we did not observe

ATM phosphorylation after treatment with doxorubicin in S3R

cells. On the other hand, a low level of the phosphorylated form of

p53 (p-p53 Ser 15) was detected in untreated, S3R cells and its

level increased after treatment with doxorubicin. Others [29]

showed impaired, but still detectable, ATM and p53 phosphor-

ylation in doxorubicin-treated NBS fibroblasts. Interestingly, in

NBS fibroblasts the p26 instead of the p70 fragment of nibrin

could be found, which doesn’t possess the ATM binding domain.

This discrepancy could be explained by the fact that p53 can be

phosphorylated on Ser 15 not only by ATM, but also by DNA-

PK, which plays a vital role in DSB repair as well as in driving cells

to apoptosis [30]. Nonetheless, the results obtained by Hou et al.

[29] allowed to conclude that NBS1 is acting upstream of ATM.

On the other hand, ATM phosphorylates nibrin at its Ser 343

residue [7]. We showed that nibrin can act both downstream and

upstream of ATM, as downregulation of nibrin affected

phosphorylation of both nibrin and ATM. These results suggested

that DDR could be compromised in cells with a diminished level

of nibrin. However, in VSMCs, in which the level of nibrin was

substantially reduced, the p53/p21 pathway was practically not

affected which suggests that in normal cells there must be a

redundancy of this protein. Surprisingly, despite the presence of

the same amount of p70-nibrin in both cell lines, the p53/p21

pathway was only activated in the S4 cells. This could imply a

failure in DDR activation downstream of ATM in the S3R cells.

However, these cells had much less ATM bound to nibrin in the

IP assay.

Moreover, we detected a higher basal level of apoptosis in

control S3R cells, but a substantially lower level of the BRCA1

protein in comparison with S4 cells in the IP assay. This indicates

that S3R cells could have a limited capacity for DNA repair what

could be reflected by a very high rate of spontaneous apoptosis in

these cells. Indeed, also the basal level of p-p53 was higher in S3R

than in S4 cells indicating p53-dependent apoptosis.

It seems that DDR can be a culprit of cell senescence, therefore

we wondered whether S3R cells would be able to senesce after

treatment with doxorubicin. Indeed, in both L5 and S4 cells we

Figure 4. Levels of nibrin, p70-nibrin, MRE11, ATM and BRCA1 in the DDR complex estimated by immunoprecipitation assay. A.Level of nibrin: wild-type (p95) and the truncated form (p70) in control (C) and doxorubicin treated (D, 1 mM/1 h) S3R, S4 and VSMCs. Expression ofnibrin was analyzed by immunoprecipitation using an anti-NBS1 antibody followed by Western blotting with anti-NBS1 (upper panel). Alternatively,IP using anti-MRE11 antibody was performed followed by WB with anti-NBS1 (lower panel). MRE11 was used as a loading control. The last lane (C IP)shows the negative IP control. Note that p95 is only present in VSMCs, in which there is no p70-nibrin. B. ATM binding to nibrin in control anddoxorubicin-treated S3R and S4 cells analyzed by immunoprecipitation using anti-NBS1 antibody (upper panel) or anti-ATM antibody (lower panel).Levels of ATM and p70 were detected by WB. Loading controls were performed in both variants of IP. C. Expression of BRCA1 in control and dox-treated S3R and S4 cells was analyzed by immunoprecipitation using anti-ATM antibody followed by WB using an anti-BRCA1 antibody. Loading andnegative IP controls were performed as above.doi:10.1371/journal.pone.0104964.g004

Figure 5. Activation of the DNA damage response pathwayupon c-irradiation. Expression of the DDR proteins analyzed byWestern blotting in control (C) and exposed to radiation (IR) S3R, S4 andL5 cells. Whole cell extracts were prepared 3 h after exposure to 4 Gy ofc-radiation, b-actin was used as a loading control.doi:10.1371/journal.pone.0104964.g005



Figure 6. The role of nibrin in doxorubicin-induced senescence of human Vascular Smooth Muscle Cells (VSMCs). Cells weretransfected with negative siRNA (2) or NBN siRNA (+) and afterwards cultured for three days in the presence of doxorubicin (100 nM). A.Downregulation of the NBS1 protein level in VSMCs using specific siRNA (60 nM). Whole cell extracts were prepared at indicated time points aftertreatment with doxorubicin. Expression of the indicated proteins was estimated by Western blotting, b-actin was used as a loading control. Theamount of the protein in cells transfected with NBN siRNA was calculated by densitometry as a fraction of that present in cells transfected with



observed the appearance of the common and widely used marker

of senescence, which is increased SA-b-Gal activity. The presence

of this marker of senescence is common in adherent cells [5]

however data concerning senescence of lymphoid cells and the

presence of this hallmark is scarce [31]. Additionally increased

activity of SA-b-Gal in the S4 cells was accompanied by a time-

dependent increase in the level of p21, which is a cdk inhibitor.

Thus, we can conclude that L5 and S4 cells, contrary to S3R cells,

are able to activate the DDR and undergo senescence. Moreover

VSMCs with a highly reduced level of nibrin were also able to

undergo senescence just like cells with the proper level of this

protein. We can speculate that there is a minimal amount of nibrin

or its truncated p70 form which is indispensable for the activation

of DDR and the subsequent induction of senescence. Interestingly,

it has been shown very recently that doxorubicin treated ATM-

deficient human fibroblasts underwent Akt-dependent SIPS

without DDR activation [32]. It seems that S3R cells are unable

to activate such a program and, most likely any senescence

pathway.

We showed that S3R cells are generally more sensitive to

doxorubicin treatment than the S4 and L5 cell lines. Also others

showed extreme variations in the propensity to undergo DNA

damage-induced apoptosis (40-fold) amongst lymphoid cells

derived from the NBS patients [33]. The authors did not find a

correlation between the propensity to undergo apoptosis and the

level of the truncated form of nibrin-p70. The mechanisms of cell

death in these cells is still awaiting elucidation.

It seems that, despite the presence of a similar level of p70-

nibrin, in the S3R and S4 cell lines, the differences in ATM

phosphorylation and its ability to bind nibrin were crucial for the

efficient activation of DDR and the induction of senescence. We

observed that some proteins which are involved in the DNA

damage/repair pathway (ATM, BRCA1) were more efficiently

recruited to the DNA damage-induced complex in S4 than in S3R

cells what might explain the differences in the cell fate after

treatment with doxorubicin.

Moreover it cannot be excluded that the described in this paper

differences in the S3R and S4 cell phenotype, may result from

genomic instability of patients with Nijmegen Breakage Syndrome

or the immortalization process. It has also been previously shown

that NBS patients with the same genotype may vary in the

phenotypic expression [34]. It is worth to note that unsupervised

clustering of whole genome gene expression arrays of S3R and S4

cells indicated that common gene expression changes, between the

two lines, also exist [35].

Acknowledgments

Experiments using flow cytometry were performed at the Laboratory of

Cytometry at the Nencki Institute of Experimental Biology. We would like

to thank Mrs. Anna Leonowicz for her skillful technical assistance.

Author Contributions

Conceived and designed the experiments: OA ABZ GM ES. Performed

the experiments: OA ABZ GM WDR AW MKK. Analyzed the data: OA

ABZ GM MMV ZK KP ES. Contributed reagents/materials/analysis

tools: MMV KP AB JKS. Contributed to the writing of the manuscript:

OA ABZ GM ES.

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